The evolution of linear chromosomes in eukaryotes poses several challenges for the natural ends of chromosomes, such as misrecognition as DNA damage sites, DNA degradation, and incomplete DNA replication. A solution to these challenges has been the formation of a protective cap structure comprising a tandem array of telomeric repeats and associated proteins. The shelterin complex protects telomeres against processing by the DNA damage repair (DDR) machinery. However, the molecular basis of how shelterin remodels telomeric DNA and distinguishes telomere termini from damaged DNA ends remains unclear. These questions can be addressed by resolving the structure of shelterin-bound telomeric DNA and investigating the dynamic interplay between shelterin and DDR signals at the telomeric terminus. Telomeric tracts shorten upon each cell division until reaching a limit at which their structural failure triggers replicative senescence. Thus, telomeres are referred to as internal biological clocks that determine the proliferative lifespan of somatic cell. In germ-line and stem cells, the loss of telomeric tracts can be reversed by telomerase, a special reverse transcriptase that adds telomeric repeats to chromosome ends. Recruitment of telomerase to the telomeric terminus and its repeat addition processivity are tightly regulated by shelterin. We currently lack an integrated understanding of telomerase's mechanochemical cycle and the specific roles that shelterin components have in modulating telomerase processivity. Further insights will be gained by real-time monitoring of telomeric repeat synthesis by telomerase at a single nucleotide resolution. The primary objective of this proposal is to develop structural and single-molecule biophysical approaches in vitro to dissect the molecular mechanism of telomere maintenance and protection. We have three specific aims: First, we will reconstitute the human shelterin complex and determine the structure of the apo and the DNA-bound complexes using negative stain electron microscopy (EM). We will then attempt to obtain a high resolution structure of the complex using cryo-EM to reveal the protein-nucleic acid interactions that mediate the protection and maintenance of telomeres. Second, we will assemble shelterin on a model telomeric DNA substrate in a flow chamber and study how shelterin remodels telomeres into their protected form using single-molecule FRET. The dynamic interplay between shelterin and telomere- end targeting proteins will be monitored at telomeric terminus. We aim to address the conceptual dilemma of how shelterin protects telomeric terminus against binding of a DDR signaling protein, RPA (replication protein A), while allowing the recruitment of telomerase. Third, using optical trapping, we aim to identify the mechanochemical transitions associated with telomeric repeat synthesis by telomerase and provide a mechanistic explanation for the enhancement of telomerase processivity by shelterin subunits.